In the mid seventies a drug design programme using the Amanita muscaria constituent muscimol (7) as a lead structure, led to the design of guvacine (23) and (R)-nipecotic acid (24) as specific GABA uptake inhibitors and the isomeric compounds isoguvacine (10) and isonipecotic acid (11) as specific GABAA receptor agonists. The availability of these compounds made it possible to study the pharmacology of the GABA uptake systems and the GABAA receptors separately. Based on extensive cellular and molecular pharmacological studies using 23, 24, and a number of mono-and bicyclic analogues, it has been demonstrated that neuronal and glial GABA transport mechanisms have dissimilar substrate specificities. With GABA transport mechanisms as pharmacological targets, strategies for pharmacological interventions with the purpose of stimulating GABA neurotransmission seem to be (1) effective blockade of neuronal as well as glial GABA uptake in order to enhance the inhibitory effects of synaptically released GABA, or (2) selective blockade of glial GABA uptake in order to increase the amount of GABA taken up into, and subsequently released from, nerve terminals. The bicyclic compound (R)-N-Me-exo-THPO (17) has recently been reported as the most selective glial GABA uptake inhibitor so far known and may be a useful tool for further elucidation of the pharmacology of GABA transporters. In recent years, a variety of lipophilic analogues of the amino acids 23 and 24 have been developed, and one of these compounds, tiagabine (49) containing (R)-nipecotic acid (24) as the GABA transport carrier-recognizing structure element, is now marketed as an antiepileptic agent.

γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA _A receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA _A receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC ₅₀ = 140 nM) over α4β(2/3)δ (EC ₅₀ = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [ ³ H]( E , RS )-(6,7,8,9-tetrahydro-5-hydroxy-5 H -benzocyclohept-6-ylidene)acetic acid showed a 39% reduction ( P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA _A receptors and postulate a role for extrasynaptic α4δ-containing GABA _A receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.